Previous genetic screens have identified six par genes in C. elegans that play essential roles in cytoplasmic partitioning and asymmetric cell divisions in the early embryo. The goal of the research described in this proposal is to determine the roles of two of the six genes par-1 and par-2. To fully understand the role of the proteins encoded by these genes, a kinase and a RING finger zinc-binding protein respectively, it is necessary to identify candidate substrates of the kinase and candidate proteins that associate with the RING finger. To identify candidates, C. elegans cDNA libraries will be screened using these two proteins as lures in the yeast two-hybrid system. If the two- hybrid approach fails, then alternate approaches including phage-based expression library screening, a biochemical affinity approach or a genetic interaction screen will be used to identify candidate PAR-1 kinase substrates and PAR-2 RING finger binding proteins. To determine which candidates are involved in par-dependent processes, the candidate gene s expression will be inhibited in vivo by injection of antisense RNA into gonads of wild-type worms. Candidates that give a Par-like mutant phenotype when antisense-inhibited are likely to be involved in a par-dependent mechanism for controlling cytoplasmic asymmetries. Positive candidates will be studied using a variety of available genetic, molecular and cell biological tools in order to determine their role in asymmetric cell divisions, differentiation and development.